In previous studies, we have obtained evidence that SSAT1 overexpression leads to total inhibition of expression of GFP reporter protein. The inhibition was at the level of translation but not at transcription, since GFP mRNA or GFP-eIF5A mRNA did not decrease upon cotransfection with SSAT1. Overexpression of SSAT1 in 293T cells by use of adenovirus resulted in a rapid depletion of spermidine and spermine, arrest in protein synthesis, inhibition of cell growth and apoptotic cell death. Addition of polyamine analogs, such as alpha-methylspermidine and dimethylspermine that are not substrates for SSAT1, restored translation and cell growth, providing evidence that depletion of polyamines caused growth arrest and cell death. Inhibition of polyamine oxidases by MDL72527 did not restore cell growth suggesting that growth arrest was not induced by oxidative stress resulting from accelerated polyamine catabolism. Annexin V staining and propidium iodide Fluorescent Activated Cell Sorter (FACS) analyses showed a clear increase in apoptosis at 48 h after AdSAT1 transduction. Nuclear fragmentation and an increase in caspase 3 activity, Poly ADP ribose polymerase (PARP) cleavage and release of cytochrome c from mitochondria into cytoplasm further suggest apoptosis by the intrinsic mitochondrial pathway in polyamine-depleted cells. Moreover, electron microscopy images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including nuclear fragmentation, cytoplasm shrinkage, mitochondrial alteration, vacuolization and membrane blebbing. The effects of control GFP adenovirus (AdGFP) transduction were minor compared to those of AdSAT1 transduction, suggesting that apoptosis was mainly caused by an over-expression of SAT1 rather than adenoviral transduction per se. The apoptosis appears to result largely from depletion of the polyamines, spermidine and spermine, as polyamine analogs that are not a substrate for SAT1 could restore growth of AdSAT1-transduced cells. These data provide strong evidence that the polyamines spermidine and spermine are vital for cell viability. In addition to the role of polycationic polyamines in general translation initiation, the polyamine spermidine is required for posttranslational modification of an essential translation factor, eIF5A. As eIF5A has been recently reported as a sequence-specific elongation factor that facilitates peptide bond formation at consecutive prolines, in analogy to its bacterial ortholog EF-P, we have searched the genome databases of Escherichia. coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus and Homo sapiens for PPP (Pro-Pro-Pro) and/or PPG (Pro-Pro-Gly)-encoding genes whose expression would depend on EF-P or eIF5A and examined the abundance and frequency of PPP or PPG motifs in both total and orthologous protein pools. The PPP and PPG motifs are rare in the bacterial proteome. However, their frequency increases dramatically in eukaryotic organisms with functional complexity, and is higher in newly derived proteins than in those orthologous proteins commonly shared in all species. Ontology classifications of S. cerevisiae and human genes encoding the highest level of polyprolines reveal their strong association with several eukaryote-specialized biological processes, including actin/cytoskeletal associated functions, RNA splicing/turnover, DNA binding/transcription and cell signaling. Of all the amino acid tandem repeats (&#8805; 3 amino acids), only the proline repeat frequency correlates with the eukaryotic hierarchy. These findings lead to a hypothesis that eIF5A and its hypusine modification pathway have emerged with the increasing demand for efficient expression of proline repeat-rich proteins in the course of metazoan evolution.